CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE

Gram Stain – SOP No: CRM-SOP 7

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Gram Stain

CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE

Title: Gram Stain SOP No: CRM-SOP: 7

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TA B L E O F C O N T E N T S

Acknowledgements 3
Introduction 5
Amendment Procedure 6
Process Flow Chart 7
1. Title 8
2. Purpose 8
3. Introduction 8
4. Scope 8
5. Staff Competency Requirements 8
6. Safety Instructions 8
7. Pre-Examination Procedures 9
7.1 Sample Type 9
7.2 Sample Collection 9
7.3 Sample Transport & Storage 9
7.4 Rejection Critiria 9
7.5 Relevant Clinical Information 9
8. Table of Media, Reagents, Materials & Equipment 10
9. Examination Procedures 10
9.1 Quality Control 10
9.2 Microscopy 11
9.3 Culture 12
9.4 Identification 12
9.5 Susceptibility Testing 12
9.6 Sample Referral 12
10. Post-Examination Procedures 12
10.1 Interpretation of Results 12
10.2 Reporting 12
10.3 Sample Retention, Storage & Disposal 13
11. Limitations and Pitfalls of the Procedure 13
12. References 14
13. Appendix 15

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 Gram Stain

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Title: Gram Stain SOP No: CRM-SOP: 7

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INTRODUCTION

The Caribbean Regional Standard Methods include a variety This initiative should enable the region to implement a
of standard, validated methods, produced as a single standardized and constructive method for ensuring that
standard operating procedure (SOP) for use in a variety of validated methods are available for the region, and that they
levels of microbiology laboratory service. It is intended that are updated as required.
these methods provide detailed instructions for microbiology
services for microbiological investigations, in order to Advantages of using regionally validated methods are to
provide accurate, reliable and reproducible results which improve quality, make better use of resources, reduce costs,
will have clinical utility. These methods may be adopted by enable central procurement & media preparation, facilitate
laboratories within the region, or adapted, provided that such staff training and transfers due to horizontal integration, a
adaptations use an evidence-based validation process. reduction in variability of service provision, an improved
quality of surveillance data, and the purchase of appropriate
These methods have been developed by the Caribbean equipment. A major advantage is that the availability of
Regional Microbiology Standard Methods Drafting Group regional standard methods would assist microbiology
(CSMDG) in response to a request by the Caribbean laboratories with documentation for accreditation
Regional Microbiology Council (CRMC), which was set up by
the CARIFORUM Project entitled ‘Strengthening of Medical Although the CSMDG has taken every care with the
Laboratories in the Caribbean’ to strengthen specifically the preparation and issue of these standard procedures, and they
microbiology services in the Caribbean Region. The Project have been validated regionally, nationally and internationally,
was initiated in response to findings which indicated that the CSMDG, or any other organization, cannot be responsible
there was an unacceptable level of error in laboratories within for the accuracy of any statement or representation made
the region. External quality assessment results revealed that or the consequences arising from the use of or alteration to
microbiology laboratories were not performing well and any information contained in them. These procedures are
feedback from the region via laboratory staff, lab managers intended solely as a resource for practicing microbiology
and directors was that they felt that guidance in microbiology professionals in the field, operating in the Caribbean region,
requirements was required. and specialist advice should be obtained where necessary.
If changes are made to the original publication, it must be
The background for this initiative is a worldwide move to made clear where changes have been made to the original
implement standards in all areas, which has now extended document. When referring to these SOPs in successive
to include medical laboratories. As tourism is so vital to documentation, the CSMDG should be acknowledged.
the region’s economy, the need for accurate diagnosis and
treatment is paramount. It was accepted that there is a
requirement for validated methods for accreditation purposes
and providing validated standard methods will assist in the
move towards accreditation.

The methods will be chosen for standardization by the

Caribbean Regional Microbiology Council, and this selection
will be based on a review of EQA results, most common and/
or critical tests. Part of the method standardization process
will be an ongoing review and amendment procedure. The
CSMDG consists of microbiology laboratory representatives
from most of the CARIFORUM countries, all of whom were
nominated to the task by the CRMC.

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE

Title: Gram Stain SOP No: CRM-SOP: 7

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AMENDMENT PROCEDURE

Controlled Document Reference CRM-SOP 7

Controlled Document Title Standard Operating Procedure for Gram Stain

Each Regional Standard Method should be reviewed annually by the Caribbean Standard Methods Drafting
Group. Any amendments should be validated and authorized by an agreed process, and referenced.

Each Regional Standard Method has an individual record of amendments. The current amendments are listed
on this page.

On issue of revised or new pages, each controlled document should be updated by the copyholder in the
laboratory.

Amendment Issue Number Insert Page Section(s) Amendment

Number / Date Discarded Issue Number involved

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PROCESS FLOW CHART

GRAM STAIN

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1. Title

Gram Stain.

2. Purpose

To ensure the correct, validated procedure is followed for processing Gram Stain, to provide accurate, reliable, reproducible
results having clinical utility.

3. Introduction

This is the most widely used bacteriological staining method, with most organisms being classified by their reaction in the
Gram Stain procedure. The microscopical examination of stained preparations enables the morphology, relative sizing, and
arrangement of microorganisms to be clearly seen, and also assists in the detection of cells, especially pus cells.

Differences in the Gram reaction is due to differences in the bacterial cell wall structure. Organisms, which stain dark purple
with crystal violet, and are not decolorized by acetone alcohol are classified as Gram-positive. Organisms which, after being
stained with crystal violet, do not retain the dye complex when decolorized with acetone alcohol, and stain red with safranin
counterstain – are classified as Gram-negative.

4. Scope

The procedure provides detailed instructions for the Gram Stain, as offered by regional laboratories. It may be adopted or
adapted by any laboratory as needed, provided that such adaptations use an evidence-based validation process.

5. Staff Competency Requirements

Laboratory personnel, trained and assessed to be competent to perform this procedure.

6. Safety Instructions

Please refer to CRM-SOP 20: Safety In The Microbiology Laboratory.

Keep all books, forms and papers away from technical work surfaces.

Level II containment; require all personal protective equipment (PPE).

All stains and reagents are for in vitro diagnostic use only.

The Gram Stain reagents:

Inflammable reagents such as acetone and alcohol should be kept tightly closed and away from sources of ignition, direct
sunlight, and kept stored in a well ventilated place.

Hands should be thoroughly washed with soap and water after handling organisms and reagents.

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7. Pre-Examination Procedures

7.1 Sample Type

Clinical samples such as body fluids, tissues, pus.

Bacterial cultures from clinical samples.

7.2 Sample Collection

All microbiological samples are collected by trained, qualified, experienced nurses or physicians using published,
acceptable guidelines.

7.3 Sample Transport & Storage

Slide holders should be used to transport clinically prepared slides.

Store prepared slides at room temperature.

7.4 Rejection Criteria

Slide exposed to contaminants (e.g. dust, chemicals) cracked or broken slides, non pre-cleaned slides, unlabeled
slides.

Name on slide does not correspond with that on the requisition form or clinical sample.

Unlabeled specimen.

Sample unaccompanied by requisition form.

Dry swab received.

Sample received in an inappropriate, non-sterile container.

7.5 Relevant Clinical Information

All requisition forms for Microbiological investigations should contain relevant clinical information such as antimicrobial
therapy, any acute or chronic infections, any clinical symptoms, patient history etc.

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8. Table of Media, Reagents, Materials and Equipment

Media Reagents Materials Equipment

N/A Crystal Violet Stain Immersion Oil Microscope

Gram’s Iodine Bunsen Burner/Spirit with oil
Acetone-Alcohol Lamp/Heating Block immersion
Safranin Microscope Slide lens
Methanol Wire Loop
Sterile Peptone
Water
0.85%sterile Saline
Tap Water
Staining rack
Control slides
Lens cleaner/paper
Blotting Paper

9. Examination Procedures

9.1 Quality Control

Please refer to CRM-SOP 18: Media Preparation and Quality Control.

Please refer to CRM-SOP 21: Quality Control of Reagents and Tests.

Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organsims.

The quality of the stain and the staining procedure must be constantly monitored.

The appearance of the stain must always be checked before use to ensure that no precipitate has formed or to ensure
that there is no contamination.

Ensure that reagents and stains are not used beyond the expiry date.

Control Strains and expected results.

S. aureus ATCC 25923 – Gram-Positive Control – dark blue/purple cocci

E. coli ATCC 25922 – Gram-Negative Control – red/pink bacilli

Positive and negative control slides should be prepared, stained & examined daily or with each stain run. In addition
they should be tested, every time a new batch of stain is prepared.

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9. Examination Procedures continued

Control results must be dated, and recorded on the appropriate forms available in the department for that purpose.

Please refer to Appendix 1 of this SOP for Gram Stain control record chart.

Results are only valid when the control slides appear as expected after staining.

9.2 Microscopy

Slide Preparation

If smears are to provide reliable information, they must not only be stained well, but should also be labeled clearly,
spread evenly, and fixed with care.

If one swab is to be used for both culture and Gram Stain, always inoculate the media before preparing the smear.

9.2.1 Label clean glass slides clearly

9.2.2 Swabs – Rub the swab over the middle surface of the slide ensuring that some of the clinical material is
transferred
9.2.3 Body Fluids – Apply the material thinly and uniformly, using a sterile wire loop or a drop from a sterile
pipette spread over the middle surface of the slide. In the case of centrifuged specimens, the smear should
be made from a drop of the well mixed sediment.
9.2.4 Bacterial isolate – Using a sterile wire loop or sterile wire, emulsify a single colony in a drop of peptone
water or 0.85% sterile saline on a clean labeled slide. Spread this emulsion evenly and thinly over
the middle surface of the slide. Allow the slide to air dry.

9.2.5 Staining Method:

a. Fix slide with methanol by flooding the slide with methanol and leaving to air dry
b. If a heating block is used, leave the slide on this for 10-15 minutes OR
c. Heat fix by quickly passing the slide-smear, face uppermost, three times through an open flame, so that
the bottom of the slide is briefly in contact with the flame. The slide should not be overheated or this may
crack the slide or alter the bacterial morphology
d. Allow the slide to cool to room temperature before staining
Flood the slide with crystal violet stain for 60 seconds
Gently wash the stain off with clean water and tip off excess water Make sure that the water runs gently
over the slide surface to avoid washing the smear off the slide.
e. Flood the slide with Gram’s iodine for 60 seconds
f. Remove the mordant (iodine) with clean water, making sure that the excess is thoroughly drained from
the slide before proceeding to the next step.
g. Decolorize with acetone alcohol briefly by holding the slide at an angle over the sink. Wash and drain
until the blue color has washed out of the smear.(30-60 seconds dependant on thickness of smear and
organism involved)
h. Wash immediately with water
i. Repeat, if necessary, until the crystal violet stain is no longer running off the slide.
j. Flood the slide with safranin counterstain for 30-60 seconds
Gently wash off the stain with clean water
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9. Examination Procedures continued

k. Wipe the back of slide clean, and air dry.

l. Examine the dried smear microscopically under the x100 oil immersion objective.
If an automated staining system is used, follow manufacturer’s instructions.

9.3 Culture

N/A

9.4 Identification

Gram-positive bacteria Dark Purple

Yeast Cells Dark Purple
Gram-negative bacteria Pale to Dark Pink/Red
Nuclei of pus cells Pink-Red
Trichomonas vaginalis Pale Pink-Red

9.5 Susceptibility Testing

N/A

9.6 Sample Referral

Slides may be referred to a senior technologist or microbiologist for review if further clarification is required.

10. Post-Examination Procedures

10.1 Interpretation of Results

• Rod - bacilli
• Round - cocci
• Pink / Red - Gram-negative
• Blue / Purple - Gram-positive

10.2 Reporting

This should include the following information:

10.2.1 The numbers of bacteria present per high power field (hpf), reported as many, moderate, few or scanty.
10.2.2 The Gram reaction of the bacteria, whether Gram-positive or Gram-negative.
10.2.3 The morphology of the bacteria, whether cocci, diplococci, rods or coccobacilli, and whether the organisms are
intracellular or extracellular.
10.2.4 The presence and number of pus cells per hpf.
10.2.5 The presence and quantity of yeast cells and epithelial cells.
10.2.6 Presence/absence of organisms/appearance indicative of Bacterial vaginosis, (Clue cells).
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10. Post-Examination Procedures continued

EXAMPLE OF REPORTING CRITERIA

CMPT Program’s recommended Gram Stain reporting criteria*

Grade Description No. per oil immersion field (x1000)

Cells Bacteria
1+ Rare <1 <1
2+ Few 1-5 2-10
3+ Moderate 6-10 11-50
4+ Many >10 >50

*Based on an average of reading more than 10 fields

10.3 Sample Retention, Storage & Disposal

Storage – Store slides in holder or cabinet for a week or until the final report has been issued.

Disposal – Place slides in biohazard sharps container, sealed when full, and send to incinerator.

REAGENT STORAGE

On receipt, the staining kit should be stored at room temperature (15-30°C), away from heat and direct sunlight, and not opened
until ready for use.

The expiry date on the product applies to unopened bottles stored as directed by the manufacturers.

The Gram iodine working solution should be used within three (3) months of making up.
Ensure that reagents, stains and media are not used beyond the expiry date.

11. Limitations and Pitfalls of the Procedure

Prolonged application of acetone-alcohol may result in over-decolonization of slide.

Overheating may cause distortion of cells and microorganism.

Blotting may remove specimen on slide.

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11. Limitations and Pitfalls of the Procedure continued

Thick smears stain poorly and reveal little details and may wash off the slide.

Gram Stain of the clinical sample provides preliminary information only and is not a substitute for culture. Culture of the
specimen must be performed to confirm identity of the causative organism and to obtain susceptibility results.

Use of an 18-24 hour culture is advisable, since older cultures of Gram-positive organisms may stain as Gram-negative or
Gram-variable.

Gram Stain morphology can be affected by the age of the isolate, autolytic enzyme systems if present, and cultures from
patients on antibiotics. (Prior treatment with antibiotics may cause bizarre morphology or Gram-positive organisms staining as
Gram-negative.)

Background material, including precipitated stain and other artifacts can also affect interpretation.

Use of an unclean or greasy slides may also affect results.

12. References

netSPEAR: Standard operating procedures for clinical specimen taking, transportation and laboratory processing.
www.netspear.org

Journal of Clinical Microbiology, November 2000, Pg. 4266 - 4268, Vol. 38, No. 11. Qualitative Gram Stain Interpretation criteria
used by MicroB Labs in Alberta, Canada. D. Church, E. Melnyk, B. Unger.

Medical Laboratory Manual for Tropical Countries. Volume II: Microbiology. Monica Cheesbrough.

Lynch’s Medical Laboratory Technology, Fourth Edition. Senior Author: Stanley S. Raphael. Saunders International Edition.

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Gram Stain

APPENDIX 1
GRAM STAIN CONTROL RESULTS

Date Batch Positive Control Negative Control Comments Sign

Staphylococcus E. coli
aureus ATCC 25923
ATCC 25922

Comments:

Reviewed and dated by:

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